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Tyrosine nitration is a protein post-translational modification that is predominantly non-enzymatic and that is observed to be increased under conditions of nitrosative stress and in numerous disease states. A small protein motif (14-18 amino acids) responsive to tyrosine nitration has been developed. In this design, nitrotyrosine replaced the conserved Glu12 of an EF-Hand metalbinding motif. Thus, the non-nitrated peptide bound terbium weakly. In contrast, tyrosine nitration resulted in a 45-fold increase in terbium affinity. NMR spectroscopy indicated direct binding of nitrotyrosine to the metal and EF-Hand-like metal contacts in this designed peptide. Nitrotyrosine is an efficient quencher of fluorescence. In order to develop a sensor of tyrosine nitration, the initial design was modified to incorporate Glu residues at EF Hand positions 9 and 16 as additional metal-binding residues, to increase the terbium affinity of the peptide with unmodified tyrosine. This peptide with tyrosine at residue 12 bound terbium and effectively sensitized terbium luminescence. Tyrosine nitration resulted in a 180-fold increase in terbium affinity (K d = 1.6 µM) and quenching of terbium luminescence. This sequence was incorporated as an encoded protein tag and applied as a turn-off fluorescent protein sensor of tyrosine nitration. The sensor was responsive to nitration by peroxynitrite, with fluorescence quenched on nitration. The greater terbium affinity upon tyrosine nitration resulted in high dynamic range and sensitivity to substoichiometric nitration. An improved approach was also developed to the synthesis of peptides containing nitrotyrosine, via the in situ silyl protection of nitrotyrosine. This work represents the first designed, encodable protein motif that is responsive to tyrosine nitration.

Cysteine sulfinic acid is a protein posttranslational modification that is formed under oxidative conditions and is regulated both enzymatically and nonenzymatically. Cysteine oxidation to the sulfinic acid has been observed broadly throughout the proteome and can induce activation or inhibition of function in proteins. Recently, wide‐scale, reversible regulation of the sulfinic acid state of cysteine within proteins was identified, posing new questions in cysteine sulfinic acid biology. Existing methods to synthesize peptides with cysteine sulfinic acid can suffer from low yield, due to the formation of side products in the disulfide, sulfenic acid, and/or sulfonic acid oxidation states. Herein, a method for the synthesis of peptides with cysteine sulfinic acids was developed, via protection of cysteine sulfinic acid as the methoxybenzyl (Mob) sulfone. Cysteine Mob sulfone was synthesized as an Fmoc amino acid in one step from the commercially available Mob‐protected Fmoc‐cysteine (Fmoc‐Cys(Mob)‐OH). This amino acid was directly incorporated into peptides via solid‐phase peptide synthesis. Alternatively, peptides were synthesized using Fmoc‐Cys(Mob)‐OH, followed by subsequent oxidation within peptides of the thioether to the Mob sulfone via H2O2 and catalytic niobium carbide. Deprotection of peptides under strongly acidic conditions (50% triflic acid, 45% trifluoroacetic acid, 5% water) generated peptides with cysteine sulfinic acid. This approach was applied to the synthesis of peptides containing cysteine sulfinic acid within diverse peptide sequence contexts.